Experimental study on pathogenesis and histomorphology of early carcinoma of the extrahepatic bile duct in the Syrian hamster.

Abstract

To elucidate the pathogenesis of carcinomas in the extrahepatic bile duct, we investigated the histomorphological characteristics of adenomas and early carcinomas induced in the extrahepatic bile duct of hamsters. Syrian hamsters underwent a cholecystoduodenostomy along with a dissection of the common duct, while also being administered N-nitrosobis(2-oxopropyl)amine (BOP). The tumors that arose from the extrahepatic bile duct included 10 adenomas and 55 early carcinomas in 56 of the 156 hamsters sacrificed. All the adenomas were found to be polypoid in shape. The early carcinomas, which were restricted within the mucosal layer of the bile duct, showed the following three different growth patterns: (1) protruding type in 41 (75%), consisting of 27 polypoid and 14 papillary tumors; (2) superficial spreading type in 9 (16%); and (3) periductal glandular type in 5 (9%). There were no depressed tumors observed. Carcinomas existing either alone or associated with adenomas were evident in 12 (22%) tumors, and 11 of these were polypoid. Atypical papillary hyperplasia within the tumor mass was noted in 22 early carcinomas (40%) and was particularly prominent in papillary type tumors. These results support the concept of an adenoma-carcinoma sequence in the majority of polypoid tumors of the extrahepatic bile duct. Atypical papillary hyperplasia might also be premalignant, and these precursor lesions should reflect the growth patterns of tumors, at least in the early stage of tumorigenesis.

Full text

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It is well known that most early gastric carcinomas
are slightly depressed in gross shape, while early car-
cinomas of the colon and rectum are usually poly-
poid. Based on clinicopathological features, at least
two different concepts of colorectal carcinogenesis
have been proposed, i.e. the adenoma-carcinoma
sequence for early polypoid carcinomas (1,2) and the
de novo pathway for flat or depressed early carcino-
mas (3-5). In conjunction with this, carcinomas of the
extrahepatic bile duct at the early stage still remain a
diagnostic challenge despite recent advances in imag-
ing modalities, and there is still little known regard-
ing early neoplastic changes in the bile duct.
In our laboratory, an experimental hamster model
for the induction of biliary carcinoma was estab-
lished using the carcinogen N-nitrosobis(2-oxo-
propyl)amine (BOP) (6). Efficient induction of carci-
noma in the extrahepatic bile duct and gallbladder is
difficult to induce experimentally, but is feasible in
this hamster model. Moreover, the induced tumors
demonstrate both morphologic and biologic resem-
blances to human biliary carcinoma. Thus, we suc-
cessfully established cancer cell lines and trans-
plantable tumors of the bile duct and gallbladder
using this hamster model (7,8). Additionally, hor-
monal effects on biliary carcinogenesis were also
investigated (9).
In the present study, the histopathological charac-
teristics of adenomas and early carcinomas of the
extrahepatic bile duct induced in hamsters were
investigated with special reference to the histogenesis
and the growth pattern of the tumors. The elucidation
of the growth form of carcinomas at the early stage
and the precursor changes in the bile duct may, there-
fore, contribute to a better understanding of this high-
ly malignant tumor.
J. Exp. Clin. Cancer Res., 24, 3, 2005
475
Experimental Study on Pathogenesis and Histomorphology of
Early Carcinoma of the Extrahepatic Bile Duct in the Syrian
Hamster
Y. Tajima, T. Tomioka, Y. Ikematsu, S. Yamanaka, T. Kuroki, T. Kitajima, K. Fukuda, N. Tsuneoka, A. Kitazato, T. Adachi and T.
Kanematsu
Dept. of Transplantation and Digestive Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
To elucidate the pathogenesis of carcinomas in the extrahepatic bile duct, we investigated the histomorphologi-
cal characteristics of adenomas and early carcinomas induced in the extrahepatic bile duct of hamsters. Syrian
hamsters underwent a cholecystoduodenostomy along with a dissection of the common duct, while also being ad-
ministered N-nitrosobis(2-oxopropyl)amine (BOP). The tumors that arose from the extrahepatic bile duct includ-
ed 10 adenomas and 55 early carcinomas in 56 of the 156 hamsters sacrificed. All the adenomas were found to
be polypoid in shape. The early carcinomas, which were restricted within the mucosal layer of the bile duct,
showed the following three different growth patterns: (1) protruding type in 41 (75%), consisting of 27 polypoid
and 14 papillary tumors; (2) superficial spreading type in 9 (16%); and (3) periductal glandular type in 5 (9%).
There were no depressed tumors observed. Carcinomas existing either alone or associated with adenomas were
evident in 12 (22%) tumors, and 11 of these were polypoid. Atypical papillary hyperplasia within the tumor mass
was noted in 22 early carcinomas (40%) and was particularly prominent in papillary type tumors. These results
support the concept of an adenoma-carcinoma sequence in the majority of polypoid tumors of the extrahepatic
bile duct. Atypical papillary hyperplasia might also be premalignant, and these precursor lesions should reflect
the growth patterns of tumors, at least in the early stage of tumorigenesis.
Key Words: Carcinoma of the extrahepatic bile duct, Pathogenesis, Adenoma, Hyperplasia, Hamster

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Materials and Methods
A total of 221 7-week-old female Syrian golden
hamsters (SLC, Inc., Shizuoka, Japan) were used. The
average weight at the time of initiaton of the experi-
ments was 110 g. Three hamsters were housed in each
plastic cage and kept under standard laboratory con-
ditions in the Laboratory Animal Center for Bio-
chemical Research at Nagasaki University Graduate
School of Biomedical Sciences. The animals were
given a standard pellet diet and water ad libitum dur-
ing the experiment. All experiments were performed
following the Guidelines for Animal Experimentation
of Nagasaki University Graduate School of Biomed-
ical Sciences.
The extrahepatic bile duct tumors were induced in
hamsters according to the previously described
method (6). Briefly, the hamsters first underwent a
cholecystoduodenostomy with dissection of the extra-
hepatic bile duct on the distal end of the common
duct. All hamsters underwent surgery and 173 of
them tolerated the procedures, while 48 animals died.
The surviving hamsters were given weekly subcuta-
neous injections of N-nitrosobis(2-oxopropyl)amine
(BOP) at a dose of 10 mg/kg body weight for 9 con-
secutive weeks. BOP administration was started four
weeks after surgery. After BOP treatment, 5 hamsters
died due to the toxicity of BOP. Twelve hamsters died
of carcinomas of the liver and/or pancreas before
being sacrificed. Finally, 156 hamsters were succes-
sively killed for pathological investigations at 2-week
intervals from 12 to 20 weeks after the initial admin-
istration of BOP. Namely, 24, 26, 33, 38 and 35 ham-
sters were sacrificed at 12, 14, 16, 18 and 20 weeks,
respectively. During the autopsy, the extrahepatic bile
duct was removed en bloc with the liver, gallbladder,
pancreas and part of the duodenum, and then all
organs were fixed in 10% buffered formalin. The
extrahepatic bile duct was cut into three blocks and
embedded so that each section contained the hepatic
ducts, common bile duct and common duct. All sec-
tions were then cut and stained with hematoxylin and
eosin (H&E).
Lesions induced in the extrahepatic bile duct were
classified in accordance with the WHO classification
of tumors in hamsters (10,11). In general, adenoma
and carcinoma were defined as lesions that showed
signs of expansive growth. Carcinomas, in contrast to
adenomas, presented signs of malignancy, such as
nuclear atypia, mitotic activities, disruption of epithe-
lial cell polarity and invasion. Hyperplastic epitheli-
um with marked papillary proliferation, pseudostrati-
fication and hyperchromatism of the nuclei was con-
sidered to indicate atypical hyperplasia.
To evaluate cell kinetic activity of normal biliary
epithelium, hyperplastic epithelium with or without
atypia, adenomas, and carcinomas, proliferating cell
nuclear antigen (PCNA) was used. Tissue sections
were cut at 4µm, mounted on glass slides coated with
5-aminoprophyltriethoxy saline, and dewaxed in
xylene. The sections were treated with microwave
heating for 5 min in phosphate-buffered saline (PBS)
at 500W. After blocking of endogenous peroxidase,
the sections were incubated with mouse monoclonal
antibodies against PCNA (clone-PC 10; DAKO,
Kyoto, Japan) at a dilution of 1:100. The cell nuclei
were counterstained with hematoxylin. The propor-
tion of labeled nuclei (labeling index; LI) was deter-
mined by counting the labeled nuclei in >1000 epithe-
lial cells of the lesions. Mann-Whitney test was used
for statistical analysis of the PCNA-LI and differ-
ences of p <0.05 were considered significant.
Results
Incidence, location and histologic findings of
induced tumors. The incidence and number of tumors
induced in the extrahepatic bile duct increased,
depending on the length of the observation period after
the administration of BOP (Table I). A total of 69
tumor nodules arose from the extrahepatic bile duct in
56 hamsters. The tumor nodules were mainly located
in the hepatic ducts and the common bile duct. Of the
69 tumors, 59 were histologically diagnosed as carci-
nomas while the remaining 10 were adenomas. Papil-
lary carcinoma was histologically evident in 36 and the
tubular type in 23. In addition, 55 out of the 59 carci-
nomas were also found, to a limited extent, within the
mucosal layer of the bile duct. Four of the adenomas
demonstrated the papillary type and six the tubular
type. Pancreatic carcinomas occurred in 118 (76%)
hamsters. The majority of these tumors were ductular
adenocarcinomas. Gallbladder carcinomas and intra-
hepatic cholangiocarcinomas developed in 114 (73%)
and 107 (69%) hamsters, respectively. Gallbladder car-
cinomas were papillary in growth form, and diagnosed
as papillary adenocarcinomas. Half of the gallbladder
carcinomas showed invasive growth into the liver.
Gross shape of adenoma and early carcinoma of
the bile duct. All the adenomas showed a polypoid
form protruding into the lumen of the bile duct
(Fig.1). The 55 early bile duct carcinomas were divid-
Y. Tajima et al.
476

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ed into three types according to the gross shape of the
tumors (Table II): (1) protruding type, in which the
tumors showed two subtypes; a, pedunculated or ses-
sile polypoid type in 27 lesions (Fig.2a); b, papillary
type growing into the lumen of the bile duct with
marked papillary projections in 14 lesions (Fig.2b);
(2) superficial type spreading along the bile duct wall
with a slight elevation of the cancerous mucosa in
nine lesions (Fig.2c); and (3) periductal glandular
type developing from the periductal glands of the bile
duct with an expansive growth within the bile duct
wall in five lesions (Fig.2d). The early bile duct car-
cinomas were thus predominantly classified as the
protruding type by gross shape. There were no ulcer-
ated or depressed tumors observed.
Coexistence of non-malignant lesions. The coexis-
tence of non-cancerous components within the tumor
mass was also examined in the 55 early bile duct car-
cinomas (Table II). In the polypoid type carcinomas,
adenomatous components were found in 11 (41%) of
the 27 lesions (Fig.3a, 3b, 3c), including two carcino-
mas within the adenoma (Fig.4a, 4b). Papillary
hyperplasia with various degrees of atypia was also
observed within six polypoid carcinomas. The
remaining polypoid carcinomas did not show any evi-
dence of either adenomatous or hyperplastic changes.
In the papillary type carcinomas, hyperplastic epithe-
lium with marked papillary proliferation, pseudostrat-
ification and hyperchromatism of the nuclei were
observed within the tumor mass in 12 (86%) of the 14
lesions (Fig.5a, 5b). In the superficial spreading type
carcinomas, neither an adenomatous component nor a
hyperplastic epithelium was noted within the tumor
mass, and atypical hyperplasia with a serrate prolifer-
ation of the epithelium was observed in the border
zone between the cancerous mucosa and non-cancer-
ous mucosa. In the periductal glandular type carcino-
mas, an adenomatous component was found in one
and hyperplastic epithelium in four. In summary, ade-
nomatous components were found within 12 (22%)
and atypical papillary hyperplasia within 22 (40%) of
the 55 early carcinomas of the extrahepatic bile duct.
Cell kinetic studies. Cell kinetic activity of the bil-
Pathogenesis of Extrahepatic Bile Duct Carcinoma
477
Table I - Incidence and pathological findings of tumors induced in the extrahepatic bile duct by N-nitrosobis(2-oxo-
propyl)amine in hamsters
Experimentala)
periods
(weeks)
No.ofb)
hamsters
examined
No.(%) of
hamsters with
EHBD tumor
No. of
EHBD
tumors
Histology
carci-
noma
Major histologic type
ade- carcinoma
noma Pap Tub
Tumor location
HDCBD CD
adenoma
Pap Tub
12
14
16
18
20
24
26
33
38
35
156
5 (21)
7 (27)
11 (33)
17 (45)
16 (46)
56 (36)
6
8
4
4
6
1
2
5
8
7
23
1
2
2
2
2
9
5
7
11
19
17
59
1
1
2
3
3
10
3
5
7
11
10
36
2
2
4
8
7
23
0
1
1
1
1
4
1
0
1
2
2
6
13
22
20
69
12
11
37 Total
a)Periods from start of N-nitrosobis(2-oxopropyl)amine treatment to sacrifice of hamsters.
b)All the hamsters underwent cholecystoduodenostomy with dissection of the distal end of the common duct.
abbreviations : EHBD, extrahepatic bile duct; HD, hepatic ducts; CBD, common bile duct; CD, common duct; Pap, papillary; Tub, tubular.
Fig. 1 - Apolypoid type adenoma in the common bile duct, show-
ing simple glandular proliferation. The individual glands
were separated from each other by a broad stroma (H&E,
x60).

Page 4

Y. Tajima et al.
478
Table II - Gross shape of early carcinomas of the extrahepatic bile duct and coexisting non-cancerous component within the
tumor mass induced in hamsters
No.of coexisting
non-cancerous components
within the tumor mass
Adenoma
Major histologic type
PapillaryGross shape of the tumors No.of lesionsTubularHyperplasia
Protruding 41
27
14
9
5
23
9
14
9
1
18
18
0
0
4
11
11
0
0
1
18
6
12
0
4
Polypoid
Papillary
Superficial spreading
Periductal glandular
Total 55 33 22 1222
Fig. 2 - (A) Apolypoid type carcinoma arising in the hepatic duct. The tumor protruded into the lumen of the bile duct with a stalk (H&E,
x20). (B) Apapillary type carcinoma in the common bile duct. The tumor grew into the lumen of the bile duct with marked pap-
illary projections (H&E, x10). (C) A superficial spreading type carcinoma in the common bile duct. Cancer cells spread super-
ficially along the mucosal layer of the bile duct. Cancer cell proliferation lacked a connective tissue stalk (H&E, x40). (D) A
periductal glandular type carcinoma in the common bile duct. The tumor grew expansively within the bile duct wall (H&E, x20).
AB
CD

Page 5

iary epithelium during biliary carcinogenesis was
investigated in 56 hamsters with extrahepatic bile
duct tumors, and the results are summarized in Table
III. Carcinomas and adenomas showed an extremely
high proliferation activity. Hyperplastic epithlium
with atypia was also confirmed to possess a high
potential for proliferation.
Discussion
The existence of the adenoma-carcinoma
sequence in tumors is well recognized throughout the
gastrointestinal tract, especially in the ampulla of
Vater (12-14) and the small intestine (15) as well as in
the colon and the rectum. In the biliary system, most
of the evidence of an adenoma-carcinoma sequence
relates to the gallbladder (16,17). Although several
investigators have reported the presence of cancerous
foci in adenoma in the intra- and extrahepatic bile
duct (18-20), the histogenesis of such bile duct carci-
nomas is still a subject of debate. In the present study,
adenomatous residue, including carcinoma in adeno-
ma, was found in 12 (22%) out of 55 early carcino-
mas of the extrahepatic bile duct. In addition, the
coexistence of an adenomatous component within the
tumor mass was particularly prominent in the poly-
poid type carcinomas in 11 (41%) out of 27 cases.
The high percentage of adenomatous residue within
the tumor mass obtained in the present study is suffi-
cient to justify application of the adenoma-carcinoma
sequence to some tumors of the extrahepatic bile
duct, particularly to polypoid type carcinomas.
Yamanaka et al. (21) and Majima et al. (22) have
investigated genetic changes in the biliary epithelium
during biliary carcinogenesis using our experimental
hamster model, and demonstrated that K-ras point
mutations were frequently detected in hyperplastic
mucosal epithelium of the bile duct as well as induced
biliary carcinomas. In the present study, cell kinetic
analysis demonstrated that hyperplastic epithelium
with atypia possessed a high potential for proliferation.
Pathogenesis of Extrahepatic Bile Duct Carcinoma
479
Fig. 3 - (A) Apolypoid type carcinoma with adenomatous residue
found in the hepatic duct (H&E, x20). (B) High magnifi-
cation of the portion of carcinoma showing well differen-
tiated tubular adenocarcinoma (H&E, x100). (C) High
magnification of the adenomatous component found at
the tip of tumor of Fig. 3a. Notice the simple glandular
proliferation with a broad stroma. The nuclei were
arranged regularly close to the basement membrane and
an abundant goblet cell metaplasia was seen (H&E,
x100).
AB
C